PROJECT SUMMARY Nucleosomes (Nucs) are the repeating units of chromatin, made up of DNA wrapped around a histone octamer. Changes in chromatin structure / function can dramatically impact downstream gene expression and cellular physiology, driving oncogenic reprogramming and cancer progression. This epigenetic regulation of chromatin is controlled by two major modifications: histone post-translational modifications (PTMs; such as histone methylation or acetylation) and DNA methylation. Recent studies show that histone and DNA methylation signaling pathways are interdependent and synergistically alter the activity of chromatin modifying/interactor proteins. Indeed, most chromatin regulators contain multiple chromatin-binding modules that interact with both DNA and histone modifications, suggesting that combinatorial interactions (via DNA and histones) are fundamental to their regulation and function in vivo. However, there are currently no commercially available research tools to biochemically profile the interplay between specific histone and DNA modifications on a single assay substrate. Here, EpiCypher is developing and commercializing high-throughput assays that leverage recombinant designer nucleosomes (dNucs) containing both histone PTMs and DNA methylation (MeDNA-dNucs). This first- to-market assay platform is highly innovative, unlocking access to epigenetic drug targets that require combinatorial nucleosome modifications to recapitulate in vivo binding specificity / enzyme activity (see UHRFI example below). In Phase I, we have developed methods to generate dNucs carrying unique DNA methylation profiles (MeDNA-dNucs) and validated these reagents using effector binding and enzymatic assays. Significantly, we discovered an optimized MeDNA-dNuc substrate for the oncogenic histone targeting ubiquitin ligase enzyme UHRF1, which is overexpressed in many cancers and a powerful indicator of poor prognosis. Indeed, the binding preference, activity, and target selectivity of UHRF1 is dramatically altered when targeting MeDNA-dNucs vs. unmodified Nucs. Here, we will develop additional disease-relevant methylated DNA species (5mC, 5hmC, 5fC, and 5caC) as we scale-up manufacturing (reduce costs, increase throughput) of a diverse set of MeDNA-dNucs for therapeutic development and discovery (Aim 1). Next, we will functionally validate our MeDNA-dNuc library using EpiCodeTM, EpiCypher?s high-throughput discovery platform (Aim 2), demonstrating how these substrates can be used for novel drug / disease biomarker discovery. This work will be performed in collaboration with Dr. Scott Rothbart, an expert in DNA / histone modification crosstalk. Finally, we will demonstrate the utility of MeDNA-dNucs for drug development by developing and validating the first HTS inhibitor assays for UHRF1 (Aim 3; binding and enzymatic assays). This platform will provide novel access to challenging (or so-called ?undruggable?) epigenetic targets as well as help decipher complex chromatin signaling interactions toward the identification of new cancer biomarkers.